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Duck plague is an acute, febrile, and septic infectious disease caused by duck plague virus (DPV), which causes serious harm to the duck industry in China. Ducks latently infected with DPV display a clinically healthy state, which is one of the epidemiological characteristics of duck plague. In the present study, to rapidly distinguish vaccine-immunized ducks from wild virus-infected ducks during production, a PCR assay based on the newly identified LORF5 fragment was developed to effectively and accurately identify viral DNA in cotton swab samples and was used to assess artificial infection models and clinical samples. The results showed that the established PCR method had good specificity and that only the virulent and attenuated DNA of duck plague virus was specifically amplified, as the results for the detection of common duck pathogens (duck hepatitis B virus, duck Tembusu virus, duck hepatitis A virus type 1, novel duck reovirus, Riemerella anatipestifer, Pasteurella multocida, and Salmonella) were negative. The amplified fragments of virulent and attenuated strains were 2,454 bp and 525 bp, and their minimum detection amounts were 0.46 pg and 46 pg, respectively. The detection rate of the virulent and attenuated DPV strains in duck oral and cloacal swabs was lower than that of the gold standard PCR method (GB-PCR, which is unable to distinguish virulent and attenuated strains), and cloacal swabs from clinically healthy ducks were more suitable for detection than oral swabs. In conclusion, the PCR assay established in the present study can be used as a simple and effective method for the clinical screening of ducks that are latently infected with virulent strains of DPV and shedding virus, which can provide technical support for the elimination of duck plague from duck farms., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

We characterized naturally occurring pigeon herpesvirus (PiHV; Columbid alphaherpesvirus 1 ) infection in domestic pigeons in California. We retrieved and analyzed 62 pathology reports produced between 1991 and 2014 at the California Animal Health and Food Safety Laboratory System. In 56 of the cases, the diagnosis of PiHV infection was established based on histopathology, either alone (44 cases) or combined with virus isolation (VI; 8), transmission electron microscopy (TEM; 3), or PCR (1); in the remaining 6 cases, the diagnosis was established based on VI (5 cases) or TEM (1) alone. PiHV infection affected 1 system in 34, 2 in 16, and ≥3 systems in 6 cases; data were not available for the remaining 6 cases. Most commonly affected was the digestive system (55 cases), followed by the respiratory tract (5) and lymphoid system (2). The liver (39 cases), crop (17), and esophagus (14) were the organs affected most commonly. Many affected cells often bore single eosinophilic intranuclear inclusion bodies. PiHV infection was a secondary diagnosis or incidental finding in 35 cases. Most (55) cases had 1 (21), or up to 4 (34), other concurrent infections; the most common concurrent infections were pigeon circoviral infection (26), trichomonosis (24), aspergillosis (11), and colibacillosis (10).

Duck plague is a disease with high morbidity and mortality rates, and it causes great losses for the duck breeding industry. Duck plague virus (DPV) is the causative agent of duck plague, and DPV UL49.5 protein (pUL49.5) is homologue of glycoprotein N (gN), which is conserved in herpesviruses. UL49.5 homologues are known to be involved in processes such as immune escape, virus assembly, viral fusion, transporter associated with antigen processing (TAP) inhibition and degradation, and maturation and incorporation of glycoprotein M. However, few studies have focused on the role of gN in the early stage of virus infection cells. In this study, we determined that DPV pUL49.5 was distributed in the cytoplasm and colocalized with the endoplasmic reticulum (ER). Moreover, we found that DPV pUL49.5 was a virion component and nonglycosylated protein. To better explore its function, BAC-DPV-ΔUL49.5 was constructed, and its attachment was only approximately 25 % of the revertant virus. Additionally, the penetration ability of BAC-DPV-ΔUL49.5 has only reached 73 % of the revertant virus. The plaque sizes produced by the UL49.5-deleted virus were approximately 58 % smaller than those produced by the revertant virus. Deleting UL49.5 mainly resulted in attachment and cell-to-cell-spread defects. Taken together, these findings suggest important roles for DPV pUL49.5 in viral attachment, penetration and spread., Competing Interests: Conflicts of interest The authors have no financial or non-financial competing interests to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Feline calicivirus (FCV) and feline herpesvirus type I (FHV-1) are the most common viral pathogens responsible for cat respiratory diseases, and coinfection with these two pathogens is often found. In veterinary clinics, the main diagnostic methods for FCV and FHV-1 are test strips and polymerase chain reaction (PCR). However, the sensitivity of test strips are not sufficient, and PCR is time-consuming. Therefore, developing a rapid and high-performance clinical diagnostic test is imperative for the prevention and treatment of these diseases. Enzymatic recombinase amplification (ERA) is an automated isothermal nucleic acid amplification technique that maintains a constant temperature, and is both rapid and highly accurate. In this study, a dual ERA method was developed using the Exo probe for a differential detection of FCV and FHV-1. This dual ERA method demonstrated high performance with the detection limit of 10 1 copies for both viruses, and no cross-reactions with feline parvovirus virus and F81 cells. To test the utility of the method for clinical applications, 50 nasopharyngeal swabs from cats with respiratory symptoms were collected and tested. The positive rates of FCV and FHV-1 were 40% (20/50, 95% confidence interval [CI], 26.4 to 54.8%) and 14% (7/50, 95% CI, 5.8 to 26.7%), respectively. The rate of coinfection with FCV and FHV-1 was 10% (5/50, 95% CI, 3.3 to 21.8%). These results were in agreement with those found using quantitative real-time PCR. Therefore, this dual ERA method is a novel and efficient clinical diagnostic tool for FCV and FHV-1 detection., (© 2023. The Author(s).)

Objective: To assess the efficacy of compounded cidofovir, famciclovir, and ganciclovir for the treatment of feline herpesvirus type 1 (FHV-1) ocular surface disease., Animals Studied: 132 shelter-housed cats qPCR positive for FHV-1., Procedures: A masked placebo-controlled study design was utilized. Animals were enrolled in one of four treatment groups: topical ocular placebo + oral placebo (n = 32), compounded cidofovir 0.5% ophthalmic solution + oral placebo (n = 32), compounded famciclovir oral solution (90 mg/kg) + topical ocular placebo (n = 32), and compounded ganciclovir 0.15% ophthalmic solution + oral placebo (n = 36). Cats were treated with each medication twice daily for 7 days and were evaluated on Day 1 and Day 8 using an ocular scoring system, body weight, and qPCR for FHV-1 viral load., Results: Cidofovir significantly decreased viral load from Day 1 to Day 8 compared with placebo (p = .024). Neither famciclovir nor ganciclovir decreased viral load compared with placebo (p = .14, p = .41). There was no significant improvement of ocular scores for any drug group compared with placebo (p = .62). In all groups, 65%-75% of cats improved from Day 1 to Day 8. Juvenile cats had a significant increase in weight gain compared with placebo for cidofovir (p = .025) and ganciclovir (p = .023). All corneal ulcers in placebo animals failed to heal whereas 77% of ulcers in antiviral group animals healed., Conclusions: Topical ophthalmic cidofovir significantly decreased ocular FHV-1 viral shedding and increased weight gain in juvenile cats. Ganciclovir increased weight gain in juvenile cats. Compounded famciclovir demonstrated limited efficacy for the treatment of FHV-1 ocular surface disease in shelter-housed cats., (© 2022 American College of Veterinary Ophthalmologists.)

Elephant endotheliotropic herpesvirus-hemorrhagic disease (EEHV-HD) is the most highly fatal infectious disease among young Asian elephants. Despite the fact that antiviral therapy has been widely used, its therapeutic outcomes remain uncertain. Additionally, the virus has yet to be successfully cultivated in vitro in the process of develop viral envelope glycoproteins for vaccine design. The present study aims to investigate and evaluate EEHV1A glycoprotein B (gB) antigenic epitopes as potential candidates for further vaccine development. Epitopes of EEHV1A-gB were employed in in silico predictions and designed by using online antigenic predicting tools. Candidate genes were then constructed, transformed and expressed in the E. coli vectors prior to examine their potential for acceleration elephant immune responses in vitro. Elephant peripheral blood mononuclear cells (PBMCs) isolated from 16 healthy juvenile Asian elephants were investigated for their proliferative capability and cytokine responses after being stimulated with EEHV1A-gB epitopes. Exposure of elephant PBMCs to 20 µg/mL of gB for 72 h resulted in a significant proliferation of CD3 + cells when compared with the control group. Furthermore, proliferation of CD3 + cells was associated with a marked up-regulation of cytokine mRNA expression, involving IL-1β, IL-8, IL-12 and IFN-γ. It remains to be determined whether these candidate EEHV1A-gB epitopes could activate immune responses in animal models or elephants in vivo. Our potentially promising results demonstrate a degree of feasibility for the use of these gB epitopes in expanding EEHV vaccine development., Competing Interests: Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fibropapillomatosis is a debilitating neoplastic disease associated with Chelonid alphaherpesvirus 5 (ChHV5) infection. We detected the Atlantic variant of ChHV5 associated with a fibropapilloma in a green turtle (Chelonia mydas) found stranded on the western coast of Rio de la Plata, Argentina. This is the southernmost registered case for the southwestern Atlantic., (© Wildlife Disease Association 2023.)

Contemporary data on equine herpesvirus-1 (EHV-1) genotype (non-neuropathogenic or N 752 , neuropathogenic or D 752 and new variant or H 752 ) in clinically diseased equids is important in order to determine the frequency of these genotypes and their association with disease expression. A total of 297 EHV-1 qPCR-positive swabs collected from 2019 to 2022 from horses with respiratory disease (EHV-1), neurological disease (equine herpesvirus-1 myeloencephalopathy [EHM]) and abortion were tested for the three different EHV-1 genotypes (N 752 , D 752 and H 752 ) using qPCR allelic discrimination assays. All submissions originated from the United States and included 257 EHV-1 cases, 35 EHM cases and 5 cases of abortion. EHV-1 qPCR-positive cases were predominantly seen during winter and spring. N 752 was the predominant genotype detected in EHV-1 cases (87.5%), EHM cases (74.3%) and abortions (80%). D 752 was detected less frequently in EHV-1 cases (9.3%) and EHM cases (25.7%), while H 752 was only detected in EHV-1 cases (3.1%). While the N 752 genotype has remained the predominant genotype affecting horses with respiratory disease and abortion, it has also become a leading genotype in cases of EHM, when compared to historical data. The new H 752 genotype, first reported in the United States in 2021, has remained confined to a cluster of geographically and temporally related outbreaks and the data showed no emerging spread of H 752 since it was first reported. While the monitoring of EHV-1 genotypes is important from a diagnostic and epidemiological standpoint, it may also help establish medical interventions and preventive protocols to reduce the risk of severe complications associated with EHV-1 infection., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Elephant endotheliotropic herpesvirus (EEHV) causes a fatal hemorrhagic disease and is a significant cause of mortality in juvenile Asian elephants. A loop-mediated isothermal amplification (LAMP) method was developed to rapidly diagnose EEHV viremia. However, extracting DNA from whole blood samples to perform LAMP hampers diagnosis in a field setting. Here, we established the Direct-LAMP method, using heparinized plasma without extracting the DNA to speed up and simplify the test. EEHV-positive specimens were tested using the Direct-LAMP. The detection limit was calculated to be 10 1.3 copies/μL using the mimetic samples, which was almost identical to the value determined in LAMP in which DNA was extracted. Hence, the Direct-LAMP provided a more rapid diagnosis to save, which could prevent elephant deaths.

Bovine herpesvirus (BoAHV) types 1 and 5 are closely-related neurotropic alpha-herpesviruses. BoAHV-1 generally causes respiratory and genital disease but can occasionally cause encephalitis. BoAHV-5 is the causative agent of non suppurative meningoencephalitis in calves. During neuroinvasion, both viruses reach the central and peripheral nervous system. While brain alterations are well-described, the changes that occur in the medulla have not been fully detailed. In this work, we integrated and analyzed the virological findings, the microscopic lesions and the changes that occur in the expression of genes related to the innate immunity, cell cycle and apoptosis in the cervical medulla of calves experimentally-infected with BoAHV-1 and BoAHV-5. This will contribute to the understanding of the differential neuropathogenesis of these alpha-herpesviruses of cattle., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interest on the research, authorship and publication of the manuscript., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Feline herpesvirus-1 (FHV-1) is responsible for approximately 50% of diagnosed viral upper respiratory tract disease in cats. The virus infects and replicates in the epithelial cells located in upper respiratory tract. Commercial vaccines do not protect cats from the infection itself or development of latency. Previously, our lab developed a cell culture model using primary feline respiratory epithelial cells (pFRECs) to study respiratory innate immunity to FHV-1 and FHV-1 deletion mutants. However, the numbers of pFRECs that can be obtained per cat is limited. To improve the usage of respiratory epithelial 3D cultures in FHV-1 research, the present study immortalized feline respiratory epithelial cells (iFRECs) and characterized them morphologically and immunologically and evaluated the response to FHV-1 infection. Immortalization was achieved by transduction with Lenti-SV40T and Lenti-HPV E6/E7. Immortalized FRECs could be successfully subcultured for >20 passages, with positive gene expression of SV40T and HPV E6/E7. Immortalized FRECs expressed similar innate immunity-associated genes compared to pFRECs, including genes of Toll-like receptors (TLR1-9), interferon induced genes (OAS1, OAS3, IFI44, IFITM1, IFIT1), chemokines (CCL2, CCL3, CXCL8), pro-inflammatory and regulatory cytokines (IL-6, IL-4, IL-5, IL-12, and IL-18), and antimicrobials (DEFβ10, DEFβ4B). Finally, FHV-1 inoculation resulted in characteristic cytopathic effects starting at 24 hpi, with more than 80% cells detached and lysed by 72 hpi. Overall FHV-1 growth kinetics in iFRECs resembled the kinetics observed in pFRECs. In conclusion, we demonstrated that iFRECs are a useful tool to study feline respiratory disease including but not limited to FHV-1., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Background: Reindeer herding and husbandry is a traditional and important livelihood in Fennoscandia, and about 200,000 semi-domesticated reindeer are herded in Finland. Climatic changes, leading to ice-locked winter pastures, and encroachment of pasture-land have led to changes in reindeer husbandry, increasing the extent of supplementary or full ration feeding, which has become very common in Finland. Keeping reindeer in corrals or gathering them at permanent feeding sites will increase nose-to-nose contact between animals and they may be exposed to poor hygienic conditions. This may impact the epidemiology of infectious diseases, such as viral infections. The aim of this study was to investigate Finnish semi-domesticated reindeer for exposure to viral pathogens. Blood samples were collected from 596 reindeer (358 calves, 238 adults) in 2015, from nine reindeer slaughterhouses, representing most of the reindeer herding regions in Finland. Plasma samples were investigated for antibodies against a selection of known and potential reindeer viral pathogens by using enzyme linked immunosorbent assays (ELISA)., Results: The screening suggested that alphaherpesvirus and gammaherpesvirus (malignant catarrhal fever virus group; MCFV) were enzootic in the reindeer population, with a seroprevalence of 46.5% (range at slaughterhouse level 28.6-64.3%) and 29.0% (range 3.5-62.2%), respectively. Whereas the seroprevalence was significantly higher for alphaherpesvirus among adult reindeer (91.2%) as compared to calves (16.8%), no age difference was revealed for antibodies against gammaherpesvirus. For alphaherpesvirus, the seroprevalence in the northernmost region, having the highest animal density (animals/km 2 ), was significantly higher (55.6%) as compared to the southernmost region (36.2%), whereas the seroprevalence pattern for gammaherpesvirus indicated the opposite, with 8.1% in the north and 50.0% in the south. Four reindeer (0.7%) had antibodies against Pestivirus, whereas no antibodies were detected against Bluetongue virus or Schmallenbergvirus., Conclusions: Alphaherpesvirus and gammaherpesvirus (MCFV) seems to be enzootic in the Finnish reindeer population, similar to other reindeer herds in Fennoscandia, whereas the exposure to Pestivirus was low compared to findings in Norway and Sweden. The ongoing changes in the reindeer herding industry necessitate knowledge on reindeer health and diseases that may impact animal welfare and health of reindeer as well as the economy of the reindeer herding industry., (© 2023. The Author(s).)

Equine herpesvirus type 1 (EHV-1) is a devastating pathogen of horses, their natural hosts, and causes fatal encephalitis in non-natural hosts. We previously demonstrated that acylation of the tegument protein UL11 is required for viral replication in cultured cells. We created a mutant virus (EHV-1 UL12 trunc UL11 G2AC7AC9A), in which glycyl and cysteinyl residues at positions 2, 7 and 9 of UL11 that are normally acylated were replaced with alanyl residues. This virus, designated the 2/7/9 mutant, has a limited-replication cycle (LRC), in which replication stops after just a few cycles. Here, we tested whether the 2/7/9 mutant could be used as a vaccine against fatal encephalitis in a mouse model. A virulence test showed that the 2/7/9 mutant was not pathogenic in mice and elicited an antibody response. We also attempted to use the 2/7/9 mutant to immunize mice against a zebra-borne EHV-1, 94-137. Two trials were conducted, each with five immunized mice, five non-immunized and five control mice. In both trials, clinical signs and fatalities were much lower in the immunized mice than in the non-immunized mice. In addition, none of the mice in either trial developed neutralizing antibodies, indicating that the immunity induced by the 2/7/9 mutant was not due to neutralizing activity. The results indicate that the 2/7/9 LRC mutant has promise as a vaccine against EHV-1 infection non-natural hosts., Competing Interests: Conflicts of interest The authors declare that there are no conflicts of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In the present research pathology and molecular diagnosis of elephant endotheliotropic herpes virus-haemorrhagic disease (EEHV-HD) among Asian elephants was studied. Out of 76 cases, 20 were positive for EEHV infection in PANPOL and POL1 based semi-nested PCR. Out of 20 samples, 10 samples were fatal cases of EEHV-HD while 10 were of either subclinical or latent infection. Acute onset haemorrhagic disease with EEHV-HD had anorexia, facial and neck swelling, cyanotic buccal mucosa and tongue, nasal and ocular discharge, and colic. The hallmark of gross finding in all cases were severe haemorrhagic lesions in the internal organs viz. cyanosis of tongue with multifocal petechial haemorrhages, diffuse epicardial and endocardial haemorrhages, swollen liver (rounded edges) with parenchymal haemorrhages, serosal and mucosal haemorrhages in gastrointestinal tract, congested kidneys with corticomedullary haemorrhages, highly congested meninges, and brain capillaries with haemorrhages. Microscopic findings in all the cases had severe vascular changes in the visceral organs. Microthrombi was present in the vasculature of tongue, heart, lung, liver, kidney, and brain. The endothelial lining of most of the blood vessels were swollen with apoptotic changes. Amphophilic to basophilic intranuclear inclusion bodies were observed in the endothelial cells. Immunostaining using anti-EEHV DNAPOL hyperimmune sera revealed intense positive signals in the endothelium of blood vessels and their walls. Quantification of viral load in necropsy tissue samples revealed highest in the heart (7.4 × 10 6 /μg of sample) and least in the brain (9 × 10 3 /μg of sample). The PCR amplicons from EEHV1 specific genes (POL1(U38) and TER were subjected to partial genome sequencing which had 99.9% similarity with the EEHV1A subtype. It was concluded that Asian elephants in India are latently infected for EEHV1 and in all the fatal EEHV-HD cases, EEHV1A subtype was the causative agent with characteristic pathomorphological changes in visceral organs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Duck plague virus (DPV) is a high-morbidity fowl alphaherpesvirus that causes septicemic lesions in various organs. Most DPV genes are conserved among herpesviruses, while a few are specific to fowl herpesviruses, including the LORF3 gene, for which there is currently no literature describing its biological properties and functions. This study first addressed whether the LORF3 protein is expressed by making specific polyclonal antibodies. We could demonstrate that DPV LORF3 is an early gene and encodes a protein involved in virion assembly, mainly localized in the nucleus of DPV-infected DEF cells. To investigate the role of this novel LORF3 protein in DPV pathogenesis, we generated a recombinant virus that lacks expression of the LORF3 protein. Our data revealed that the LORF3 protein is not essential for viral replication but contributes to DPV replication in vitro and in vivo and promotes duck plague disease morbidity and mortality. Interestingly, deletion of the LORF3 protein abolished thymus atrophy in DPV-vaccinated ducks. In conclusion, this study revealed the expression of avian herpesviruses-specific genes and unraveled the role of the early protein LORF3 in the pathogenesis of DPV. IMPORTANCE DPV is a highly lethal alphaherpesvirus that causes duck plague in birds of the order Anseriformes. The virus has caused huge economic losses to the poultry industry due to high morbidity and mortality and the cost of vaccination. DPV encodes 78 open reading frames (ORFs), and these genes are involved in various processes of the viral life cycle. Functional characterization of DPV genes is important for understanding the complex viral life cycle and DPV pathogenesis. Here, we identified a novel protein encoded by LORF3 , and our data suggest that the LORF3 protein is involved in the occurrence and development of duck plague.

Equid alphaherpesvirus 1 (EHV-1) infection causes significant health problems in equines. The EHV-1 infection leads to abortion storm in mares, respiratory disease and myeloencephalopathy. Despite the wide use of vaccines, the outbreaks of EHV-1 infections keep occurring globally, suggesting the need for the development of improved vaccines. Gene deletion attenuated mutant viruses could be a good candidate for the development of modified live vaccines. Here, we report the generation of mutant EHV-1 by deleting virulence (glycoprotein E & internal repeat 6; IR6) and immune evasive (pUL43 & pUL56) associated genes either individually or in combinations; and comprehensive evaluation of mutants through in vitro characterization followed by in vivo study in murine model to adjudge the attenuation of the virus and immune responses generated by mutants vis-à-vis wild type (wt) virus. The EHV-1 mutants with deletion of IR6 and gE genes (vToH-DMV) and four genes (i.e., gE, IR6, pUL43 and pUL56) (vToH-QMV) revealed a significant reduction in plaque size with minimal loss in replication efficiency in comparison to the wt virus. Further, in vivo studies showed virus attenuation adjudged through significant reduction in clinical signs, weight loss, gross and histopathological lesions in comparison to wt virus also revealed improved immune responses estimated through serum neutralization and flow cytometric analysis of CD4 + and CD8 + cell populations. Thus it can be concluded that EHV-1 mutants viz. vToH-DMV and vToH-QMV (novel combination) are promising vaccine candidates and qualify to be studied for adjudging the protective efficacy with wt virus challenge., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Ocular herpes simplex type 1 (HSV-1) infections can trigger conjunctivitis, keratitis, uveitis, and occasionally retinitis, and is a major cause of blindness worldwide. The infections are lifelong and can often recrudesce during periods of stress or immune suppression. Currently HSV-1 infections of the eye are managed primarily with anti-viral eye drops, which require frequent administration, can cause irritation, and may take weeks for full resolution of symptoms. We therefore evaluated the effectiveness of an ocular immune activating nanoparticle eye drop as a novel approach to treating HSV-1 infection, using a cat feline herpesvirus -1 (FHV-1) ocular infection model. In vitro studies demonstrated significant induction of both type I and II interferon responses by the liposome-dual TLR 3/9 agonist nanoparticles, along with suppression of FHV-1 replication. In cats with naturally occurring eye infections either proven or suspected to involve FHV-1, ocular nanoparticle treated animals experienced resolution of signs within several days of treatment, including resolution of keratitis and corneal ulcers. In a cat model of recrudescent FHV-1 infection, cats treated twice daily with immune nanoparticle eye drops experienced significant lessening of ocular signs of infection and significantly fewer episodes of viral shedding compared to control cats. Treatment was well-tolerated by all cats, without signs of drug-induced ocular irritation. We concluded therefore that non-specific ocular immunotherapy offers significant promise as a novel approach to treatment of HSV-1 and FHV-1 ocular infections., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: SD, KW and LC currently hold US patent "Compositions and methods for enhancing innate immunity in a subject for treatment of infections and cancer and other acute and chronic conditions of the eye" SD and LC and ML also hold equity in a start-up company (Laporte Immunotherapeutics) that is developing the ocular immunotherapy technology, (Copyright: © 2023 Lappin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cyprinid herpesvirus 3 (CyHV-3) can cause severe disease in koi and common carp ( Cyprinus carpio ). Currently, no effective treatment is available against CyHV-3 infection in koi. Both LSD1 and JMJD2 are histone demethylases (HD) and are critical for immediate-early (IE) gene activation essential for lytic herpesvirus replication. OG-L002 and ML324 are newly discovered specific inhibitors of LSD1 and JMJD2, respectively. Here, HD inhibitors were compared with acyclovir (ACV) against CyHV-3 infection in vitro and in vivo. ML324, at 20-50 µM, can completely block ~1 × 10 3 PFU CyHV-3 replication in vitro, while OG-L002 at 20 µM and 50 µM can produce 96% and 98% inhibition, respectively. Only about 94% inhibition of ~1 × 10 3 PFU CyHV-3 replication was observed in cells treated with ACV at 50 µM. As expected, CyHV-3 IE gene transcription of ORF139 and ORF155 was blocked within 72 h post-infection (hpi) in the presence of 20 µM ML324. No detectable cytotoxicity was observed in KF-1 or CCB cells treated for 24 h with 1 to 50 µM ML324. A significant reduction of CyHV-3 replication was observed in ~6-month-old infected koi treated with 20 µM ML324 in an immersion bath for 3-4 h at 1-, 3-, and 5-days post-infection compared to the control and ACV treatments. Under heat stress, 50-70% of 3-4-month-old koi survived CyHV-3 infection when they were treated daily with 20 µM ML324 in an immersion bath for 3-4 h within the first 5 d post-infection (dpi), compared to 11-19% and 22-27% of koi in the control and ACV treatments, respectively. Our study demonstrates that ML324 has the potential to be used against CyHV-3 infection in koi.

Elephant endotheliotropic herpesvirus (EEHV) is the causative agent of EEHV-hemorrhagic disease (EEHV-HD) in elephants worldwide. This disease is highly virulent and a predominant cause of fatalities in young Asian elephants. Rapid diagnosis and aggressive therapies have been determined to be a key strategy in the successful treatment of this disease. Herein, we have developed the immunochromatographic strip test for EEHV detection. Accordingly, 31.2 kDa of partial EEHV DNA polymerase (DNApol) protein was expressed in Escherichia coli and used to generate rabbit polyclonal anti-EEHV DNApol antibodies. These were then used to develop an ICS test for EEHV antigen detection using the double-antibody sandwich colloidal gold method. Anti-EEHV DNApol antibodies conjugated with 40 nm colloidal gold solution were used as a detector, while rabbit anti-EEHV DNApol and goat anti-rabbit IgG antibodies immobilized on the nitrocellulose membrane were used as the test and control lines, respectively. The test had a detection limit of 1.25 × 10 5 viral genome copies (vgc)/mL of EEHV obtained from blood samples. Moreover, no specialized equipment or laboratory infrastructure was required in the administration of this test. This developed ICS test for EEHV antigen detection can be used in field application for the rapid detection of EEHV in resource-limited environments., Competing Interests: Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Elephant endotheliotropic herpesvirus (EEHV) infection can cause acute, often fatal, EEHV hemorrhagic disease in free-ranging and human-managed Asian elephants (Elephas maximus) and human-managed African elephants (Loxodonta africana). However, significant knowledge gaps exist pertaining to the presence of EEHV in free-ranging African elephant populations. We retrospectively screened 142 opportunistically collected samples (blood, n=98; bronchoalveolar lavage (BAL) fluid, n=21; trunk wash (TW) fluid, n=23) obtained between 2010 and 2020 from 98 free-ranging African elephants in the Kruger National Park, South Africa, for the presence of different EEHVs, as well as determining the real-time quantitative PCR positivity rate in this population. With the use of validated, previously published DNA extraction and real-time quantitative PCR protocols provided by the National Elephant Herpesvirus Laboratory (Washington, DC, USA), EEHV was detected in nine male African elephants from samples collected in 2011 (n=1), 2013 (n=1), 2018 (n=2), 2019 (n=4), and 2020 (n=1). Viral detection was more common in respiratory compared with blood samples. Six elephants tested positive for EEHV2 subtype (blood, n=2; BAL, n=3; TW, n=2), including one individual that tested positive on matched respiratory samples (BAL and TW). Four elephants tested positive for EEHV3-4-7 (blood, n=1; BAL, n=2; TW, n=1), whereas EEHV6 was not detected in any of the study animals. One elephant tested positive for both EEHV2 and EEHV3-4-7 in the same BAL sample. Even though the levels of viremia varied between 158 and 1,292 viral genome equivalents/mL blood and viral shedding of EEHV2 and EEHV3-4-7 was detected in respiratory samples, no clinical signs were observed in these apparently healthy elephants. These findings are consistent with reports of asymptomatic EEHV infection in human-managed African elephants., (© Wildlife Disease Association 2023.)

Infections caused by bovine herpesvirus 1 (BoHV-1) remain a serious global issue to the health and welfare of the bovine industry. Monitoring of neutralizing antibodies is essential not only for epidemic diagnosis, but also to assess vaccination efficacy. In this study, we generated a neutralizing monoclonal antibody, termed as 3F8, targeting glycoprotein D (gD) of BoHV-1. This monoclonal antibody could neutralize BoHV-1 with a 50% inhibitory concentration (IC 50 ) of 37.82 ng/mL. Furthermore, 3F8 could inhibit BoHV-1 infection and cell-to-cell spread at the prebinding stage. A blocking enzyme-linked immunosorbent assay (ELISA) for detecting neutralizing antibodies against BoHV-1 was then developed based on 3F8 and protein gD generated using a baculovirus expression system. The sensitivity and specificity of the test were estimated to be 94.59% and 93.42%, respectively. A significant correlation (R 2  = 0.9583, p < 0.01) was observed between the results obtained with the blocking ELISA and a virus neutralization test, which suggested that the blocking ELISA could detect neutralizing antibodies against BoHV-1. A serological survey was carried out in the dairy farms in Beijing district using 3F8-based blocking ELISA to monitor the annual neutralization antibody against BoHV-1 during 2012-2020. It revealed that the dairy farms in Beijing were at high risk of BoHV-1 infection during 2012-2017 but were protected since 2018 upon implementation of an immunization program. Our results demonstrated that this assay is suitable for BoHV-1 surveillance and vaccination efficacy in cattle as a replacement for the virus neutralization test. KEY POINTS: • Prevention of BoHV-1 infection requires the monitoring of neutralizing antibodies. • A blocking ELISA for the neutralizing antibody was developed based on mAb 3F8 against BoHV-1 gD. • It can replace the labor-intensive and time-consuming viral neutralizing tests., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Hemorrhagic disease due to elephant endotheliotropic herpesvirus infection (EEHV-HD) is an important cause of calf mortality in managed and free-ranging Asian ( Elephas maximus ) and African elephant ( Loxodonta spp.) populations. Consequently, infection has profound implications for elephant population growth and sustainability. The mechanisms of disease caused by EEHV (i.e., infection, dissemination, shedding, latency) are relatively undefined, in part because of a lack of robust validated assays for detecting viral gene products in relevant samples. To address this issue, we used RNAscope ® in situ hybridization (ISH) based on EEHV1A DNA polymerase and terminase genes to detect EEHV1A RNA in archival formalin-fixed, paraffin-embedded Asian elephant heart and tongue from PCR-confirmed cases ( n = 4) of EEHV-HD and Asian elephants ( n = 2) that died from other causes. EEHV1A-positive cases had positive hybridization signal in endothelial cell nuclei of both tissues for both DNA polymerase and terminase. EEHV-negative cases lacked signal. In positive cases, the number of positive nuclei was manually assessed to provide an estimate of the viral load and compare sensitivity of the two probes. In all cases, heart had greater signal than tongue for both probes (Wilcoxon rank test; P ≤ 0.01). Overall, terminase hybridization signal was greater than DNA polymerase signal (Wilcoxon rank test; P ≤ 0.01). Results indicate RNAscope ISH is a valuable tool for detection of EEHV in archival samples and for confirming infection. Additionally, the terminase gene is the optimal target and heart is preferable to tongue for detection in cases of EEHV-HD. Results will inform future investigations of viral tropism in EEHV-HD cases due to EEHV1A.

Bovine herpesvirus 1 (BoHV-1) is a significant risk factor for the bovine respiratory disease complex (BRDC), a severe disease causing great economic losses to the cattle industry worldwide. Previous studies have reported that both phospholipase C-γ1 (PLC-γ1) and β-catenin are activated during BoHV-1 infection for efficient replication. However, the interplay between PLC-γ1 and β-catenin as a consequence of virus infection remains to be elucidated. Here, we reported that PLC-γ1 interacted with β-catenin, which was enhanced following virus infection. PLC-γ1-specific inhibitor, U73122, significantly reduced the mRNA levels of β-catenin in BoHV-1-infected cells; however, the steady-state protein levels were not affected due to the virus infection. Interestingly, the treatment of virus-infected cells with U73122 reduced the accumulation of activated β-catenin [p-β-catenin(S552)] in fractions of the cytoplasmic membrane as that observed with the treatment of methyl-β-cyclodextrin (MβCD), which can disrupt cytoplasmic membrane structure via sequestering cholesterol. Nucleus accumulation of p-β-catenin(S552) was increased following U73122 treatment in virus-infected cells. In addition, the association of p-β-catenin(S552) with cytoplasmic membrane induced by the virus infection was significantly disrupted by the treatment of U73122 and MβCD. These data indicated that the PLC-γ1 signaling is potentially involved in the regulation of β-catenin signaling stimulated by BoHV-1 infection partially via affecting the subcellular localization of p-β-catenin(S552)., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Four Indian ringneck parakeets ( Psittacula krameri ; syn. ringneck parrots or rose-ringed parakeets) were submitted by 2 private owners for autopsy following a history of dyspnea and death. Gross findings were varied and included thickening of the left caudal thoracic air sac, white spots throughout the liver, mild dilation of the proventriculus, coelomic effusion, splenomegaly, and pulmonary congestion and edema. Microscopically, the submitted parakeets had significant lesions in the lower respiratory tract, including necrotizing bronchitis, parabronchitis, and interstitial pneumonia with numerous syncytia containing eosinophilic intranuclear inclusions. Electron microscopy of the lungs was compatible with a herpesviral infection and Psittacid alphaherpesvirus 5 (PsAHV5) was detected via PCR and sequencing. There has been inconsistent terminology used with Psittacid alphaherpesvirus 3 and PsAHV5; we attempt here to clarify the reported history of these viruses.

Cyprinid herpesvirus-2 (CyHV-2) is an important virus that causes herpesviral hematopoietic necrosis disease (HVHND) leading to huge economic losses in goldfish (Carassius auratus). However, until now no proper prophylactic measure or treatment is available for CyHV-2 infection in goldfish. Hence, in this experiment, we developed a heat-inactivated CyHV-2 vaccine and evaluated its performance in goldfish. Initially, CyHV-2 was propagated in the fantail goldfish fin (FtGF) cell line and the titer of the viral inoculum was 10 7.8 TCID 50 /ml. Subsequently, various temperatures (40 °C, 50 °C, 60 °C, 70 °C, and 80 °C) were evaluated to achieve the complete inactivation of CyHV-2. Only the viral inoculum inactivated at 80 °C for 1 h did not show any cytopathic effect in the FtGF cell line after five blind passages. Hence the heat-inactivated CyHV-2 vaccine developed at 80 °C was further used for immunization trials in goldfish. The experimental goldfish were intraperitoneally immunized with 300 μL of the heat-inactivated CyHV-2 vaccine. Subsequently, the kidney and spleen tissues were sampled at various time points post-vaccination (6th hr, 2nd day, 4th day, 6th day, 10th day, 16th day, and 30th day) to evaluate the expression of immune genes (IL-12, IL-10, IFN-γ, CD8, and CD4). A significant upregulation of immune genes was observed at various time points in the kidney and spleen tissue of the vaccinated goldfish. Furthermore, in order to study the efficacy of the vaccine, the experimental fish were challenged with CyHV-2 (10 7.8 TCID 50 /ml) after the 30th day post-vaccination. The survival of the fish in the vaccine group (86.7%) was significantly higher compared to the non-vaccinated group (20%). Moreover, the relative percentage survival of the vaccinated group was 83.34%. In spite of the single dose, the heat-killed vaccine developed in the present study elicited the immune response and offered better protection in goldfish against CyHV-2. However, further large-scale field performance evaluation studies are necessary to develop this vaccine on a commercial scale., Competing Interests: Conflict of interest disclosure The authors have no conflicts of interest to declare., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Equine herpesvirus 1 (EHV1) infection is a global health problem in equines and the virus is responsible for abortions, respiratory disease and myeloencephalitis in horses. Disease management requires proper biosecurity and immunoprophylactic measures. Vaccines strengthening both arms of immunity are essential for proper control and there has been a continuous focus in this area for generation of better vaccines. Here we report construction of bacterial artificial chromosome (BAC) clone of EHV-1 strain Tohana for mutagenesis of the virus and generation of gE gene deletion mutant EHV1. The BAC clone was generated by inserting the mini-F plasmid replacing ORF71 of EHV1 and transforming into E. coli for generation of EHV1-BAC. The infectious virus was regenerated from EHV-1 BAC DNA in RK13 cells. To check utility of EHV1-BAC, we have generated mutant EHV1 by deleting the virulence-associated gE gene. The mutant virus (vToHΔgE) showed significantly reduced plaque size without affecting replication efficiency. Pathological evaluation of lesions in BALB/c mice infected with vToHΔgE revealed reduction in clinical signs and pathology in comparison to the wild-type virus. Generation of infectious BAC of EHV1 and its usage in construction of attenuated viruses shows potential of the technology for development of indigenous modified live vaccine for EHV1. Keywords: quine herpesvirus 1; bacterial artificial chromosome (BAC); mutation; glycoprotein E; vaccine.

Bovine herpesvirus type 1 (BoHV 1) is a major bovine pathogen spreading worldwide and causing extensive damage to the livestock industry. BoHV causes respiratory, genital, and neurological disorders. A cross-sectional study was performed for the first time to estimate the seroreactivity to BoHV 1 and related risk factors among Iran's central desert dairy cattle. A total of 800 blood samples was randomly collected from 76 unvaccinated herds. Samples were tested with an indirect enzyme-linked immunosorbent assay (ELISA) commercial kit to detect BoHV 1 antibodies. The logistic regression model was used to analyze the data. BoHV 1 seroreactivity at animal and herd levels was 50% and 65%, respectively. Herd size was recognized as a risk factor (OR = 2.65, CI = 1.61-4.37) for seroreactivity to BoHV using GLM (p < 0.05). The high prevalence of BoHV 1 antibodies in the study area indicates the need to implement educational programs on the importance of the disease and design methods to control and prevent virus distribution., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Equid gammaherpesvirus 2 (EHV-2) and 5 (EHV-5) are widely distributed in the equines. Although their pathogenic potential is not yet fully understood, they appear to play a role in disease patterns like equine multinodular pulmonary fibrosis. In this study, a multiplex real-time PCR (rtPCR) was designed to detect DNA of the glycoprotein H (EHV-2) and E11 gene (EHV-5). Analytical specificity was determined by testing DNA of other herpesviruses by SYBR Green rtPCR and melting curve analysis, as well as Sanger sequencing of positive field samples. Analytical sensitivity was assessed by standard curve generation of serial plasmid dilutions containing the respective target gene. Melting curves and BLAST analysis of the sequences indicated specific detection of the viruses. The lower limit of detection of the singleplex rtPCR was 40 and 29 DNA copies per reaction for EHV-2 and EHV-5, respectively. Comparison of the Ct values of a selection of positive field samples showed only minimal differences between the singleplex and the multiplex assay. The here described multiplex rtPCR protocol allows sensitive and specific detection of EHV-2 and EHV-5. It represents a convenient and rapid tool for future studies to investigate the clinical relevance of EHV-2 and EHV-5 in more detail., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Avian infectious laryngotracheitis (ILT) is a respiratory disease that causes severe economic losses in the poultry industry, mainly due to high morbidity and mortality and reduced egg production. Molecular characterization was performed on samples collected from flocks in the Brazilian States of São Paulo, Pernambuco, and Minas Gerais during 2015 and 2016 that presented clinical signs of respiratory disease. End-point PCR was used for viral detection, and DNA sequencing was used for differentiation of vaccine and field strains. Molecular analysis based on the infected cell protein (ICP4) gene separated four of the nine samples together with previous Brazilian isolates (São Paulo and Minas Gerais), one sample was grouped on the same branch as Minas Gerais strains (along with another related sample), one sample was separately branched but still related to the tissue culture origin (TCO) vaccine strain, and two samples were grouped on the same branch as the TCO vaccine strain. Molecular analysis of the thymidine kinase (TK) gene showed the existence of strains of both high and low virulence. The characterization of two fragments of the ICP4 gene and a fragment of the TK gene in this study suggested that the virus circulating in Guatapará, as well as those in Barretos and Itanhandu, that is causing respiratory problems in birds is a highly virulent field strain. The clinical signs point to a TCO vaccine strain that most likely underwent some reversal event and is a latent reactivated infection., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Cyprinid herpesvirus 3 has a worldwide distribution and presents high mortality rates in species of Cyprinus carpio, causing serious economic loss to the global aquaculture industry. The description of this infection in other ornamental fish species is still limited. For this purpose, 100 ornamental fish from 24 different species were tested by PCR for Cyprinid hespesvirus 3 and the positive samples represented 6% of the tested samples. Phylogenetic reconstruction, based on the Thymidine Kinase gene, revealed the existence of two distinct clades. One clade grouped a Brazilian sample with European and Asian genotypes of CyHV-3 and a second clade, containing only Brazilian sequences described in this study. All of the Brazilian sequences showed identity values greater than 97.7% when compared to each other. This is the first report of the occurrence of Cyprinid herpesvirus 3 in ornamental fish species in Brazil. These results in association with further studies of viral isolation and characterization can help in establishing effective surveillance and disease control program., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Equid Herpesvirus Myeloencephalopathy (EHM) is a multifactorial disease following an EHV-1 infection in Equidae. We investigated a total of 589 horses on 13 premises in Europe in search of risk factors for the development of EHM. We found that fever ( p < 0.001), increasing age ( p = 0.032), and female sex ( p = 0.042) were risk factors for EHM in a logistic mixed model. Some breeds had a decreased risk to develop EHM compared to others (Shetland and Welsh ponies; p = 0.017; p = 0.031), and fewer EHV-1-vaccinated horses were affected by EHM compared to unvaccinated horses ( p = 0.02). Data evaluation was complex due to high variability between outbreaks with regards to construction and environment; viral characteristics and the virus's transmissibility were affected by operational management. This study confirms earlier suspected host-specific risk factors, and our data support the benefit of high vaccine coverage at high-traffic boarding facilities.

Objective: To evaluate the effect of a multidose acyclovir protocol on koi herpesvirus (KHV) viral load and mortality in a cohabitation challenge., Animals: 180 koi fish., Procedures: Forty fish (shedders) were immersed in a 0.5 KHV plaque-forming units/mL static bath for 8 hours. Mock shedders were treated similarly but exposed to cell culture media. KHV shedders were then transferred into 8 tanks (5 shedders per tank) containing 10 naïve fish (cohabitants) each. Fish in the acyclovir group (AT) received a 10 mg/kg acyclovir intracoelomic injection 1, 3, and 6 days after the first confirmed KHV mortality. Positive controls (PC) were treated similarly but received sterile saline injections. Negative controls (NC) were exposed to mock shedders. Morbidity and mortality were evaluated daily for 50 days post-challenge. Quantitative PCR was used to determine viral load in the gill biopsies of shedders and cohabitants collected at days 19 (T1), 22 (T2), 25 (T3), 34 (T4), and 50 (T5) post-challenge., Results: Survival curves analyzed by the Gehan-Breslow-Wilcoxon method revealed a delayed onset of mortalities and a significantly lower KHV load at T2 and T3 detected in AT cohabitant fish (P = .042) compared to PC group. However, there were no significant differences in overall mortality or viral loads at T5., Clinical Relevance: The acyclovir protocol used in this study did not control viral infection or mortality at the end of the 50-day trial. Shorter intervals between injections could improve outcomes, but the additional stress inflicted by handling should be considered. Exploring other therapeutic alternatives and doses is warranted.

Bovine alphaherpesviruses (BoHV) 1 and 5 share many genetic and structural characteristics; however, they differ in their ability to cause encephalitis. Previous research suggests that this difference might be caused by a differential modulation of apoptosis. In this study, we analyzed the mRNA expression of Bax, Bcl-2, Fas and caspases 3 and 8 in neural tissue sections of BoHV-1 and BoHV-5 experimentally-infected cattle. Overall, Fas and caspase 3 mRNA was up-regulated during BoHV-5 acute infection, latency, and reactivation. Conversely, caspase 3 mRNA levels increased only in the olfactory cortex during BoHV-1 acute infection, and it was down-regulated during reactivation, while Fas was only up-regulated during BoHV-1 acute infection and latency. Moreover, Bax/Bcl-2 ratio was lower than 1 during BoHV-1 acute infection except in the trigeminal ganglion, whereas many brain regions exhibit a ratio higher than 1 during BoHV-5 acute infection and reactivation. In summary, our findings suggest that during acute infection and reactivation, BoHV-5 induces a pro-apoptotic condition that could partially justify its increased ability to cause neurological damage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Infections of Cyprinid herpesvirus 2 in goldfish and farmed crucian carp (Carassius auratus gibelio) are still an urgent problem worldwide. Detection and prevention are necessary for the control of haematopoietic necrosis disease caused by CyHV-2. Although many sensitive molecular diagnostic methods have been developed, effective immunodiagnosis and neutralization approaches based on monoclonal antibodies (MAbs) against CyHV-2 are still important to CyHV-2 study. In this experiment, purified CyHV-2 was used as antigens to produce monoclonal antibodies (Mabs). Six Mabs bound to different proteins were selected by Dot-blot screening and Western-blot analysis, and no one had cross-reactivity with closely related koi herpesvirus. Among them, Mabs 2E1-B10, 1F5-A3 and 4C4-A7 belonged to IgG 1 isotype, while other three Mabs 3G9-B11, 3B4-G5 and 4F4-B7 belonged to IgM isotype. These six Mabs all could specifically detect CyHV-2 in CyHV-2 infected caudal fin of Carassius auratus gibelio (GiCF) cells by immunofluorescence assays. Then, the neutralization ability was tested in vitro, and the result showed that all six Mabs can attenuate CPE by CyHV-2 in vitro among which 2E1-B10 had the best neutralization ability. The virus proteins recognized by these six Mabs were identified by mass spectrometry identification, and the result showed they probably were ORF88, ORF55R, ORF115 and ORF151R. This study is the first to prepare Mabs by purifying CyHV-2, which will provide a practical basis for the in-depth study of CyHV-2 virus., (© 2022 John Wiley & Sons Ltd.)

Bovine Herpesvirus Type 1 (BoHV-1) infection causes infectious bovine rhinotracheitis and genital disease in cattle, with significant economic and welfare impacts. However, the role of cellular host factors during viral replication remains poorly characterised. A previously performed genome-wide CRISPR knockout screen identified pro- and antiviral host factors acting during BoHV-1 replication. Herein we validate a pro-viral role for a candidate from this screen: the cellular protein tetracopeptide repeat protein 4 (TTC4). We show that TTC4 transcript production is upregulated during BoHV-1 infection. Depletion of TTC4 protein impairs BoHV-1 protein production but does not reduce production of infectious virions, whereas overexpression of exogenous TTC4 results in a significant increase in production of infectious BoHV-1 virions. TTC4 itself is poorly characterized (especially in the context of virus infection), but is a known co-chaperone of heat shock protein 90 (HSP90). HSP90 has a well-characterized pro-viral role during the replication of diverse herpesviruses, and we therefore hypothesized that HSP90 is also pro-viral for BoHV-1. Drug-mediated inhibition of HSP90 using geldanamycin at sub-cytotoxic concentrations inhibited both BoHV-1 protein production and viral genome replication, indicating a pro-viral role for HSP90 during BoHV-1 infection. Our data demonstrates pro-viral roles for both TTC4 and HSP90 during BoHV-1 replication; possibly, interactions between these two proteins are required for optimal BoHV-1 replication, or the two proteins may have independent pro-viral roles., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Feline calicivirus (FCV), Feline panleukopenia virus (FPV), and Feline herpesvirus type I (FHV-1) are the three most common pathogens in cats, and also are the main pathogens leading to the death of kittens. Here, by a combination of gold nanoparticles and conventional PCR, we established a novel triple NanoPCR molecular detection method for clinical detection., Results: The triple NanoPCR molecular detection is able to detect 2.97 × 10 1 copies/μL FCV recombinant copies plasmid per reaction, 2.64 × 10 4 copies/μL FPV recombinant copies plasmid per reaction, and 2.85copies/μL FHV-1 recombinant copies plasmid per reaction at the same time. The sensitivity of each plasmid is 100 times, 10 times, and 100 times higher than conventional PCR, respectively. The clinical results showed that among the 38 samples, the positive rates of FCV, FPV, and FHV-1 in a NanoPCR test were 63.16, 31.58, and 60.53%, while in a conventional PCR were 39.47, 18.42, and 34.21%., Conclusions: In this report, it is the first time that NanoPCR assays are applied in the detection of FCV, FPV, and FHV-1 as well. This sensitive and specific NanoPCR assay can be widely used in clinical diagnosis and field monitoring of FCV, FPV, and FHV-1 infections., (© 2022. The Author(s).)

Infectious laryngotracheitis (ILT), caused by infectious laryngotracheitis virus (ILTV), occurs sporadically in poultry flocks in Canada. Live attenuated chicken embryo origin (CEO) vaccines are being used routinely to prevent and control ILTV infections. However, ILT outbreaks still occur since vaccine strains could revert to virulence in the field. In this study, 7 Canadian ILTV isolates linked to ILT outbreaks across different time in Eastern Canada (Ontario; ON and Quebec; QC) were whole genome sequenced. Phylogenetic analysis confirmed the close relationship between the ON isolates and the CEO vaccines, whereas the QC isolates clustered with strains previously known as CEO revertant and wild-type ILTVs. Recombination network analysis of ILTV sequences revealed clear evidence of historical recombination between ILTV strains circulating in Canada and other geographical regions. The comparison of ON CEO clustered and QC CEO revertant clustered isolates with the LT Blen® CEO vaccine reference sequence showed amino acid differences in 5 and 12 open reading frames (ORFs), respectively. Similar analysis revealed amino acid differences in 32 ORFs in QC wild-type isolates. Compared to all CEO vaccine strains in the public domain, the QC wild-type isolates showed 15 unique mutational sites leading to amino acid changes in 13 ORFs. Our outcomes add to the knowledge of the molecular mechanisms behind ILTV genetic variance and provide genetic markers between wild-type and vaccine strains., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Population-level sampling based on qPCR detection of infectious laryngotracheitis virus (ILTV) in poultry dust can be used to assess ILT vaccination outcomes following mass administration in drinking water. We report on the field application of this approach to assess the success of vaccine administration and its use in ILT outbreak control in meat chickens. In Study 1, dust samples were collected from 26 meat chicken flocks at 0, 4, 7, 14, and 21 days post drinking water vaccination (DPV) given between 7 to 13 days of age with the Serva or A20 live attenuated ILT vaccines. Unexpectedly, ILTV DNA was detected in dust samples collected prior to vaccination in 22/26 flocks. Typing revealed that the detected ILTV was different from the vaccine virus. To determine whether the detected ILTV DNA was from active infection or carryover of a noninfectious virus, Study 2 was implemented in 14 additional flocks with dust samples collected at 0, 7, 14, and 21 DPV and tracheal swabs collected from 15 birds/flock at 0 and 21 DPV. The results indicated that there was active infection with ILTV in those flocks before vaccination. This approach contributed to a statewide control program resulting in the eradication of ILT from South Australia as confirmed by negative ILTV test results for dust samples from 50 flocks and the absence of clinical ILT. These findings show that ILTV infection prior to vaccination is common in outbreak situations and that dust samples must be collected at 0 and 7 DPV for meaningful interpretation of vaccination outcomes and ILTV status. Comparatively low-cost dust testing during an outbreak, coupled with typing information, greatly assisted with decision making and control strategies during a major outbreak, including confirmation of the absence of infection in the final stages.

Infectious laryngotracheitis (ILT) is an economically crucial respiratory disease of poultry that affects the industry worldwide. Vaccination is the principal tool in the control of the disease outbreak. In an earlier study, we comprehensively characterized the circulating strains in Egypt and identified both CEO-like and recombinant strains are dominant. Herein, we investigated the pathogenicity of two virulent strains representing the CEO-like (Sharkia&#95;2018) and recombinant strain (Qalubia&#95;2018). Additionally, we evaluated the efficacy of different commercial vaccines (HVT-LT, CEO, and TCO) against the two isolates in terms of the histopathological lesion scores and the viral (gC) gene load. A total of 270 White Leghorn-specific pathogen-free male chicks were divided into nine groups of 30 birds, each housed in separate isolators. Birds were distributed as follows; one group was non-vaccinated, non-challenged, and served as a negative control. Two groups were non-vaccinated and infected with the two isolates of interest and served as a positive control to test the pathogenicity. Six groups were vaccinated and challenged; two groups were vaccinated with vector vaccine at one day old. The other four groups were vaccinated with either the CEO- or TCO- vaccine (two groups each) at four weeks of age. Three weeks after vaccination, birds were infected with the virulent ILTV isolates. The larynx, trachea, and harderian gland samples were taken at 1, 3, and 7 days post-infection for histopathological lesion score and molecular detection. Notably, The recombinant strain was more virulent and pathogenic than CEO-like ILTV strains. Moreover, the TCO vaccine was less immunogenic than the vector and CEO vaccines., (© 2022. The Author(s).)

Cyprinid herpesvirus 2 (CyHV-2) is a causative factor of herpesviral hematopoietic necrosis (HVHN) in farmed crucian carp ( Carassius carassius ) and goldfish ( Carassius auratus ). In this study, we analyzed the genomic characteristics of a new strain, CyHV-2 SH-01, isolated during outbreaks in crucian carp at a local fish farm near Shanghai, China. CyHV-2 SH-01 exhibited a high sensitivity to goldfish and crucian carp in our previous research. The complete genome of SH-01 is 290,428 bp with 154 potential open reading frames (ORFs) and terminal repeat (TR) regions at both ends. Compared to the sequenced genomes of other CyHVs, Carassius auratus herpesvirus (CaHV) and Anguillid herpesvirus 1 (AngHV-1), several variations were found in SH-01, including nucleotide mutations, deletions, and insertions, as well as gene duplications, rearrangements, and horizontal transfers. Overall, the genome of SH-01 shares 99.60% of its identity with that of ST-J1. Genomic collinearity analysis showed that SH-01 has a high degree of collinearity with another three CyHV-2 isolates, and it is generally closely related to CaHV, CyHV-1, and CyHV-3, although it contains many differences in locally collinear blocks (LCBs). The lowest degree of collinearity was found with AngHV-1, despite some homologous LCBs, indicating that they are evolutionarily the most distantly related. The results provide new clues to better understand the CyHV-2 genome through sequencing and sequence mining.

Cook Inlet beluga whales (CIBs) Delphinapterus leucas are Critically Endangered and genetically distinct from other beluga populations in Alaska. CIBs are exposed to numerous natural and anthropogenic sources of mortality and morbidity. This study describes congenital defects observed in 2 CIB calves. The first case, an aborted fetus, was characterized by lack of a peduncle and flukes, anorectal and genitourinary dysgenesis, and probable biliary dysplasia. The second case, a male calf, had a perineal groove defect and suspected secondary peritonitis; it also had a systemic herpesvirus infection. Further studies are needed to determine if such defects are due to genetic mutation, infectious diseases, nutritional imbalances, or contaminant exposure.

Crucian carp ( Carassius auratus ) is one of the major freshwater species and is also a common food fish in China. Recently, Carassius auratus herpesvirus (CaHV) could induce fatal viral disease with high mortality of crucian carp, which had caused huge economic losses. In this study, we described a rapid and simple recombinase-aid amplification (RAA) assay coupled with lateral flow dipstick (LFD), which could achieve sensitive diagnosis of tumor necrosis factor receptor (TNFR) of CaHV within 35 min at 40°C. Our RAA-LFD method had a satisfactory detection limit of 100 gene copies per reaction, which was 100-fold more sensitive than traditional PCR. In addition, no cross-reaction was observed with other viral pathogens, including koi herpesvirus (KHV), cyprinid herpesvirus 2 (CyHV-2), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). Furthermore, the overall cost of the method was cut in half compared to previous studies. In conclusion, RAA-LFD assay is therefore, a promising alternative for point-of-care testing (POCT) of CaHV, which is feasible and of certain value in application of aquatic disease control., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer WH declared a shared affiliation, with no collaboration, with several of the authors, LG, YZ, DX, XL, and ML, to the handling editor at the time of review., (Copyright © 2022 Gui, Zhao, Xu, Li, Luo, Zhou and Li.)

Infectious laryngotracheitis (ILT) is an acute respiratory disease in chickens that is a serious threat to poultry-producing countries worldwide. In the present study, we isolated and characterized infectious laryngotracheitis (ILTV) virus isolates by sequencing and restriction fragment length polymorphism analysis of PCR-amplified products (PCR-RFLP). A total of 26 ILTV outbreaks were investigated that occurred between 2019 and 2020 in flocks that had not been vaccinated against ILTV. ILTV was isolated by cultivating tracheal samples in embryonated chicken eggs, which showed multiple opaque pock lesions and thickening of the chorioallantoic membrane after 120 hours of infection. The ILTV isolates were identified and characterized by PCR and sequencing a portion of the ICP4 and TK genes. Phylogenetic analysis based on the ICP4 region showed that the sequences clustered with chicken-embryo-origin vaccine-like strains. Sequence analysis of the ICP4 region differentiated chicken-embryo-origin (CEO), tissue-culture-origin (TCO), and field ILTV strains, with significant differences in nucleotide and amino acid sequences. Furthermore, PCR-RFLP analysis of the TK gene showed that the patterns were identical to those obtained with low-virulence and vaccine strains. In conclusion, sequencing of a portion of the ICP4 region of ILTV allowed differentiation of ILTV field, CEO, and TCO vaccine strains. In this study, CEO-vaccine-like strains were found to be the cause of ILTV outbreaks between 2019 and 2020 in Tamil Nadu in southern India., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Equine rhinopneumonitis is an acute, highly contagious disease found virtually worldwide. The purpose of the studies presented in this paper is to develop a technology for the manufacture of a cell-derived equine rhinopneumonitis vaccine, as well as to assess the safety and immunogenicity of the newly developed vaccine in laboratory animals model. The object of the studies was the AK-2011 strain isolated from the horses suffering from rhinopneumonitis during an outbreak of abortions. The viability of the AK-2011 strain was assessed using a continuous line of calf trachea cells, a continuous line of calf kidney cells, a continuous line of sheep kidney cells, a continuous line of bovine kidney cells, a continuous line of green monkey kidney cells, a continuous line of Syrian hamster kidney cells, a primary trypsinized culture of horse kidney cells grown in tubes and flasks and the AK-2011 laboratory strain of equine rhinopneumonitis virus with biological activity of 6.0 lg TCID 50/cm 3 . Sequencing and polymerase chain reaction analysis were performed. The virus isolated from the ORF68 gene in Kazakhstan appeared to be the most similar to the T-953 and 2222-03 strains isolated in the USA and Australia, respectively, in terms of phylogenetics. As to primary infections, cytopathic effects (CPEs) induced by the AK-2011 virus stain (dilution 10 1 ) in calf trachea and horse kidney cell cultures were stable from the first to tenth passages, with biological activity of 5.75-6.00 lg TCID 50/cm 3 . CPEs caused by the virus were apparent on days 2-3, further developed intensively and extended to 60-80% of the cell monolayer on days 5-7. The vaccine results can be used to immunize horses on farms against rhinopneumonia, and horses should be immunized twice with an interval of 2-3 months., (© 2022 Wiley-VCH GmbH.)

A six-year study on water buffaloes from the Campania Region (Southern Italy) was conducted to evaluate the presence of bovine/bubaline herpesviruses in cases of abortion. A total of 244 buffalo foetuses were analysed by real-time PCR to detect the presence of: bovine alphaherpesvirus 1(BoHV-1), bubaline alphaherpesvirus 1 (BuHV-1), bovine alphaherpesvirus 2 (BuHV-2), and bovine gammaherpesvirus 4 (BoHV-4). The foetuses of 14 water buffaloes that showed abortions were positive for BuHV-1 (4 animals) and/or BoHV-4 (11 animals), with one of these cases showing co-infection with BuHV-1 and BoHV-4. This study reports the first identification of BoHV-4 in water buffaloes. Cases of abortion were analysed using both molecular and cultural assays for the presence of other pathogens. In nearly all the abortion cases positive for BoHV-4, the virus was identified as a co-infecting agent together with other microorganisms, whereas in two abortion cases, it was the only pathogen found., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Protection against infectious diseases can be obtained with vaccines generating immunogenic response through a combination of humoral and cellular immunity. In this study haematological and serum protein electrophoretic profiles of horses vaccinated against herpesvirus 1 (EHV-1) and 4 (EHV-4) were evaluated. Blood samples were collected from 16 horses before (T0), after 24h, 48h, 72h, 1st week, 2nd week and 3rd week (T1I, T2I, T3I, T7I, T14I and T21I) from the first EHV vaccine-dose administration as well as before (TPRE II ), and after 24h, 48h, 72h, 1st week, 2nd week, 3rd week and 4th week (T1II, T2II, T3II, T7II, T14II, T21II and T28II) from the EHV vaccine-booster. Total leukocyte values increased at T1I, T1II, T3II and T28II compared to T0 (P < .01). Higher lymphocytes and lower neutrophils values were found after first vaccine-dose and vaccine-booster administration compared to the T0 (P < .01). Monocytes showed higher values at T14II than T0 (P < .01). Higher serum values of total proteins, α1-, α-2-, β1-, β2- and γ-globulins were found in horses after first vaccine-dose and vaccine-booster administration (P < .01). Gathered results suggest that horses vaccinated against EHV1 and EHV-4 exhibited a dynamic change of WBC, lymphocytes, neutrophils and monocytes. Moreover, the analysis of serum electrophoresis pattern suggested that EHV vaccination induced the development of inflammation and antibody response in vaccinated horses as highlighted by the increase of α-, β- and γ-globulin fractions. These changes probably reflect the systemic immunological adaptation of animals to EHV vaccine., (Copyright © 2022. Published by Elsevier Inc.)

Elephant endotheliotropic herpesviruses (EEHVs) have co-existed with elephants for millions of years, yet may cause fatal haemorrhagic disease (EEHV-HD), typically in elephants between 1 and 10 years of age. EEHV is omnipresent in (sub)adult elephants, and young elephants with low EEHV-specific antibody levels are at risk for EEHV-HD, suggesting that fatal disease may occur due to an insufficiently controlled primary infection. To further address this hypothesis, sera of three large elephant cohorts were subjected to a multiple EEHV species ELISA: (I) 96 Asian elephants between 0 and 57 years, including 13 EEHV-HD fatalities, from European zoo herds typically sized five to six elephants, (II) a herd of 64 orphaned elephants aged 0-15 years at the Elephant Transit Home in Sri Lanka and (III) 31 elephants aged 8-63 years, part of a large herd of 93 elephants at Pinnawala Elephant Orphanage, Sri Lanka. All Sri Lankan elephants showed high EEHV-specific antibody levels regardless of their age. While antibody levels of most European zoo elephants were comparable to those of Sri Lankan elephants, the average antibody level of the European juveniles (1-5 years of age) was significantly lower than those of age-matched Sri Lankan individuals. Moreover, the European juveniles showed a gradual decrease between 1 and 4 years of age, to be attributed to waning maternal antibodies. Maintenance of high levels of antibodies in spite of waning maternal antibodies in young Sri Lankan elephants is likely due to the larger herd size that increases the likelihood of contact with EEHV-shedding elephants. Together with the observation that low levels of EEHV-specific antibodies correlate with increased numbers of EEHV-HD fatalities, these results suggest that infection in presence of high maternal antibody levels may protect calves from developing EEHV-HD, while at the same time activating an immune response protective in future encounters with this virus., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Cyprinid herpesvirus 2 (CyHV-2) has caused great losses to the gibel carp (Carassius auratus gibelio ) industry. Previous studies showed that certain DNA viruses can encode circular RNAs (circRNAs) to regulate virus infection, which provides new clues for the treatment of viral disease. Whether CyHV-2 can encode circRNAs is still unknown. Here, 10 CyHV-2-derived circRNAs were identified, and the function of circ-udg, a circRNA derived from the CyHV-2 uracil DNA glycosylase ( udg ) gene, was studied. Although the expression level of circ-udg was lower than that of the parental gene, udg , its expression level was elevated in tandem with the proliferation of CyHV-2 and udg . In vitro experiments confirmed that circ-udg could promote the proliferation of CyHV-2. Moreover, circ-udg could encode a truncated UDG protein consisting of 147-amino-acid residues (termed circ-udg-P147). Both UDG and circ-udg-P147 were found to promote CyHV-2 proliferation, but the promoting effect of circ-udg on CyHV-2 proliferation was attenuated after circ-udg lost the ability to encode circ-udg-P147. Also, circ-udg-P147 could not change the transcription level of the udg gene. Interestingly, the UDG protein level was increased by circ-udg-P147. These results deepen the understanding of the genetic information carried by the genome of CyHV-2 and provide a new target for the treatment of gibel carp bleeding disease caused by CyHV-2. IMPORTANCE The outbreak of C. auratus gibelio gill hemorrhagic disease caused by CyHV-2 brought great losses to the gibel carp industry. Therefore, exploring the interaction between CyHV-2 and host and the molecular mechanism of viral infection is of great significance in preventing and treating the gibel carp gill hemorrhagic disease. Although some progress has been made in the study of CyHV-2, the mechanism of interaction between CyHV-2 and crucian carp is still unclear. In this study, we found that CyHV-2 can encode circRNA to regulate virus replication. Our study provides novel information on CyHV-2 functional genomics, a reference for research into the circRNA of other viruses, and theoretical guidance for preventing and treating gibel carp bleeding disease.

Asian elephants are an endangered species facing many threats, including severe hemorrhagic disease (HD) caused by the elephant endotheliotropic herpesvirus (EEHV). EEHV-HD is the leading cause of death in captive juvenile Asian elephants in North America and Europe, and also affects elephants in their natural range countries. Significant challenges exist for successful treatment of EEHV-HD, which include timely recognition of disease onset and limited availability of highly effective treatment options. To address this problem, our goal is to prevent lethal disease in young elephants by developing a vaccine that elicits robust and durable humoral and cell-mediated immunity against EEHV. EEHV glycoprotein B (gB) is a major target for cellular and humoral immunity in elephants previously exposed to EEHV. Therefore, we generated a vaccine containing recombinant EEHV1A gB together with a liposome formulated TLR-4 and saponin combination adjuvant (SLA-LSQ). CD-1 mice that received one or two vaccinations with the vaccine elicited significant anti-gB antibody and polyfunctional CD4 + and CD8 + T cell responses, while no adverse effects of vaccination were observed. Overall, our findings demonstrate that an adjuvanted gB protein subunit vaccine stimulates robust humoral and cell-mediated immune responses and supports its potential use in elephants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)